Jomha N M, Law G K, McGann L E
Department of Surgery, University of Alberta, #1002, 8215-112St, College Plaza, Edmonton, AB Canada T6G 2C8.
Cell Tissue Bank. 2006;7(1):55-60. doi: 10.1007/s10561-005-4521-x.
Transplantation of osteochondral allograft tissue can treat large joint defects but is limited by tissue availability, surgical timing, and infectious disease transmission. Fresh allografts perform the best but requirements for infectious disease testing delay the procedure with subsequent decrease in cell viability and function. Hypothermic storage at lower temperatures can extend tissue banking time without loss of cell viability and, therefore, increase the supply of allograft tissue. This study investigated the effects of different cryoprotectant solutions on intact AC at various subzero temperatures.
10 mm porcine osteochondral dowels were immersed for 30 minutes in various combinations of solutions [(XVIVO, propylene glycol (51% w/w), sucrose (46% w/w)] cooled to various subzero temperatures (-10, -15, and -20 degrees C), and held for 30 min. After warming, 70 mum slices were stained with membrane integrity dyes, viewed under fluorescence microscopy and cell recovery calculated relative to fresh controls.
Results demonstrated excellent cell recovery (>75%) at -10 degrees C provided ice did not form. Excellent cell recovery (>70%) occurred at -15 degrees C in solutions containing 51% propylene glycol but formation of extra-matrix ice in other solutions resulted in significant cell loss. All groups had <6% cell recovery at -20 degrees C and propylene glycol did not provide a protective effect even though extra-matrix ice did not form
These results suggest that extra-matrix ice plays an important role in cell damage during cryopreservation. Excellent cell recovery can be obtained after storage at subzero temperatures if ice does not form. Hypothermic preservation at high subzero temperatures may extend AC storage time in tissue banks compared to current techniques.
同种异体骨软骨移植组织可用于治疗大关节缺损，但受组织可用性、手术时机和传染病传播的限制。新鲜同种异体移植物效果最佳，但传染病检测要求会延迟手术，进而导致细胞活力和功能下降。在较低温度下进行低温保存可延长组织储存时间，且不会损失细胞活力，从而增加同种异体移植组织的供应。本研究调查了不同冷冻保护剂溶液在不同零下温度下对完整骨软骨的影响。
将10毫米的猪骨软骨柱浸入冷却至不同零下温度（-10、-15和-20摄氏度）的各种溶液组合（XVIVO、丙二醇（51%重量/重量）、蔗糖（46%重量/重量））中30分钟，然后保持30分钟。复温后，将70微米厚的切片用膜完整性染料染色，在荧光显微镜下观察，并相对于新鲜对照计算细胞回收率。
结果表明，在-10摄氏度且未形成冰的情况下，细胞回收率极佳（>75%）。在含有51%丙二醇的溶液中，-15摄氏度时细胞回收率极佳（>70%），但其他溶液中细胞外基质冰的形成导致显著的细胞损失。在-20摄氏度时，所有组的细胞回收率均<6%，即使未形成细胞外基质冰，丙二醇也未提供保护作用。
这些结果表明，细胞外基质冰在冷冻保存过程中的细胞损伤中起重要作用。如果不形成冰，在零下温度保存后可获得极佳的细胞回收率。与当前技术相比，在较高零下温度下进行低温保存可能会延长骨软骨在组织库中的储存时间。
